Genome-wide ribonucleotide detection in Archaea

Genome integrity is constantly challenged by the incorporation of ribonucleotides (rNMPs) during DNA synthesis. Covalently linked single and several consecutive rNMPs occur in the genome of a number of organisms. They are mainly introduced by DNA polymerases during DNA replication and repair. In general, cells evolved ribonucleases H (RNases H) specialized in the removal of rNMPs from DNA to avoid any detrimental consequences on genome stability. Here, we describe the involvement of types 1 and/or 2 RNases H in processing embedded rNMPs in the genome of two archaeal species Haloferax volcanii and Thermococcus barophilus. Using combined approaches that include alkaline DNA fragmentation, high-throughput ribose-seq DNA sequencing and nucleotide pool quantification, the distribution, identity, level and sequence context of genomic rNMPs are reported and discussed regards to the intracellular balances of dNTPs and rNTPs. Our results confirm the predominant role of type 2 RNase H in the removal of genomic rNMPs. They also reveal rNMP-base compositions, densities, locations, and variations of surrounding bases at rNMP-embedment for each mutant. The cellular roles of the different RNases H in processing rNMPs in the genome of Archaea are discussed.